U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 SCREENING-LEVEL HAZARD CHARACTERIZATION Acetyl Tributyl Citrate (CASRN 77-90-7) The High Production Volume (HPV) Challenge Program1 was conceived as a voluntary initiative aimed at developing and making publicly available screening-level health and environmental effects information on chemicals manufactured in or imported into the United States in quantities greater than one million pounds per year. In the Challenge Program, producers and importers of HPV chemicals voluntarily sponsored chemicals; sponsorship entailed the identification and initial assessment of the adequacy of existing toxicity data/information, conducting new testing if adequate data did not exist, and making both new and existing data and information available to the public. Each complete data submission contains data on 18 internationally agreed to "SIDS" (Screening Information Data Setl1'2) endpoints that are screening-level indicators of potential hazards (toxicity) for humans or the environment. The Environmental Protection Agency's Office of Pollution Prevention and Toxics (OPPT) is evaluating the data submitted in the HPV Challenge Program on approximately 1400 sponsored chemicals by developing hazard characterizations (HCs). These HCs consist of an evaluation of the quality and completeness of the data set provided in the Challenge Program submissions. They are not intended to be definitive statements regarding the possibility of unreasonable risk of injury to health or the environment. The evaluation is performed according to established EPA guidance2'3 and is based primarily on hazard data provided by sponsors; however, in preparing the hazard characterization, EPA considered its own comments and public comments on the original submission as well as the sponsor's responses to comments and revisions made to the submission. In order to determine whether any new hazard information was developed since the time of the HPV submission, a search of the following databases was made from one year prior to the date of the HPV Challenge submission to the present: (ChemID to locate available data sources including Medline/PubMed, Toxline, HSDB, IRIS, NTP, AT SDR, IARC, EXTOXNET, EPA SRS, etc.), STN/CAS online databases (Registry file for locators, ChemAbs for toxicology data, RTECS, Merck, etc.), Science Direct and ECHA4. OPPT's focus on these specific sources is based on their being of high quality, highly relevant to hazard characterization, and publicly available. OPPT does not develop HCs for those HPV chemicals which have already been assessed internationally through the HPV program of the Organization for Economic Cooperation and Development (OECD) and for which Screening Initial Data Set (SIDS) Initial Assessment Reports (SIAR) and SIDS Initial Assessment Profiles (SIAP) are available. These documents are presented in an international forum that involves review and endorsement by governmental authorities around the world. OPPT is an active participant in these meetings and accepts these documents as reliable screening-level hazard assessments. 1 U.S. EPA. High Production Volume (HPV) Challenge Program; http://www.epa.gov/chemrtk/index.htm. 2 U.S. EPA. HPV Challenge Program - Information Sources; http://www.epa.gov/chemrtk/pubs/general/guidocs.htm. 3 U.S. EPA. Risk Assessment Guidelines; http://cfpub.epa.gov/ncea/raf/rafguid.cfm. 4 European Chemicals Agency, http://echa.europa.eu. ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 These hazard characterizations are technical documents intended to inform subsequent decisions and actions by OPPT. Accordingly, the documents are not written with the goal of informing the general public. However, they do provide a vehicle for public access to a concise assessment of the raw technical data on HPV chemicals and provide information previously not readily available to the public. 2 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Chemical Abstract Service Registry Number (CASRN) Chemical Abstract Index Name 77-90-7 1,2,3-Propanetricarboxylic acid, 2-(acetyloxy)-, 1,2,3-tributyl ester Structural Formula O ^"3 SMILES: 0=C(0C(C(=0)0CCCC)(CC(=Q)0CCCC)CC(=0)0CCCC)C Summary Acetyl tributyl citrate is a liquid with moderate vapor pressure and moderate water solubility. It is expected to have low mobility in soil. Volatilization is expected to be high, but adsorption to soil and sediment may attenuate the rate of volatilization. The rate of hydrolysis is negligible. The rate of atmospheric photooxidation is moderate. Acetyl tributyl citrate is readily biodegradable. Acetyl tributyl citrate is expected to have low persistence (PI) and low bioaccumulation potential (Bl). Acute oral toxicity of acetyl tributyl citrate in cats and rats is low. In a 90-day repeated-dose oral dietary study in rats, decreased body weight and organ weight changes were observed at 1000 mg/kg-bw/day; the NOAEL is 300 mg/kg-bw/day. In a combined repeated- dose/reproductive/developmental toxicity study in rats, organ weight and histopathological changes were observed in adults at 1000 mg/kg-bw/day; the NOAEL is 300 mg/kg-bw/day. In a 2-generation reproductive toxicity study in rats, reduced body weight was observed in Flmales at 300 mg/kg-bw/day; the NOAEL for systemic toxicity is 100 mg/kg-bw/day. In the same study, no other treatment related effects were observed; the NOAEL for reproductive toxicity is 1000 mg/kg-bw/day, the highest dose tested. In the combined repeated- dose/reproductive/developmental toxicity study in rats previously described, histopathological changes were observed in the liver of adult males at 300 mg/kg-bw/day; the NOAEL for systemic toxicity is 100 mg/kg-bw/day. In the same study, decreased litter size and decreased number of implantations were observed at 1000 mg/kg-bw/day; the NOAEL for reproductive and developmental toxicity is 300 mg/kg-bw/day. The NOAEL for maternal toxicity is 1000 mg/kg-bw/day (highest dose tested). Acetyl tributyl citrate did not induce gene mutations in bacteria or mammalian cells in vitro and did not induce chromosomal aberrations in mammalian cells in vitro. For acetyl tributyl citrate, the 96-h LCso values for fish range from 38 to 60 mg/L, the 48-h ECso value for aquatic invertebrates is 7.8 mg/L and the 72-h EC50 values for aquatic plants are 11.5 mg/L for biomass and 74.4mg/L for growth rate, respectively. The chronic 21-d EC50 values for aquatic invertebrates are > 1.11 mg/L (survival) and >1.11 mg/L (reproduction). No data gaps were identified under the HPV Challenge Program. 3 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 The sponsor, Morflex, Inc., submitted a Test Plan and Robust Summaries to EPA for acetyl tributyl citrate (CASRN 77-90-7; CA Index name: 1,2,3-propanetricarboxylic acid, 2- (acetyloxy)-tributyl ester) on January 12, 2004. EPA posted the submission on the ChemRTK HPV Challenge website on March 4, 2004 (http://www.epa.gov/oppt/chemrtk/pubs/summaries/acetlcit/cl5025tc.htm). EPA comments on the original submission were posted to the website on March 8, 2005. Public comments were also received and posted to the website. 1. Chemical Identity 1.1 Identification and Purity Acetyl tributyl citrate is a citric acid, trialkyl ester, acetate. Test substance purity when noted in the Robust Summaries was > 98%. 1.2 Physical-Chemical Properties Acetyl tributyl citrate is a liquid with moderate vapor pressure and moderate water solubility. The physical-chemical properties of acetyl tributyl citrate are summarized in Table 1. Table 1. Physical-Chemical Properties of CASRN 77-90-71 Property Value CASRN 77-90-7 Molecular Weight 402.5 Physical State Liquid Melting Point -59°C (measured pour point) Boiling Point 326°C (measured) Vapor Pressure 0.052 mm Hg at 20°C (measured) Dissociation Constant (pKa) Not applicable Henry's Law Constant 5.5><10"3 atm-m3/mole(estimated)2 Water Solubility <100 mg/L at 25°C (measured); 5 mg/L at 25°C (measured)3 Log Kow 4.92 at 22°C (measured) 1 The Morflex Inc. 2003. Test Plan and Robust Summary for Acetyltributyl citrate. Available online at http://www.epa.gov/chemrtk/pubs/summaries/acetlcit/cl5025tc.htm as of April 30, 2012. 2U.S. EPA. 2012. Estimation Programs Interface Suite™ for Microsoft® Windows, v4.10. U.S. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as of April 30, 2012. 3SRC. 2012. The Physical Properties Database (PHYSPROP). Syracuse, NY: Syracuse Research Corporation. Available online at http://www.svrres.com/esc/phvsprop.htm as of April 30, 2012. 4 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 2. General Information on Exposure 2.1 Production Volume and Use Pattern Acetyl tributyl citrate had an aggregated production and/or import volume in the United States between 1 and 10 million pounds during calendar year 2005. Non-confidential information in the IUR indicated that the industrial processing and uses of the chemical include other basic organic chemical manufacturing as corrosion inhibitors and anti- scaling agents and functional fluids; resin and synthetic rubber manufacturing, and other chemical and allied products merchant wholesalers as functional fluids; adhesive manufacturing, other basic inorganic chemical manufacturing, other plastic product manufacturing, paints and coating manufacturing, pharmaceutical and medicine manufacturing, plastic packaging materials and unlaminated film and sheet manufacturing, and paint ink manufacturing as "other." Non- confidential commercial and consumer uses of this chemical include metal products, rubber and plastic products and "other." 2.2 Environmental Exposure and Fate Acetyl tributyl citrate is expected to have low mobility in soil. It achieved 82% of its theoretical biochemical oxygen demand (BOD) using an activated sludge and the modified MITI (OECD 301C) test after 28 days and is considered readily biodegradable. It was shown to biodegrade extensively in several other biodegradation studies and simulation tests, suggesting that it is not persistent in the environment. Volatilization is expected to be high, but adsorption to soil and sediment may attenuate the rate of volatilization. The rate of hydrolysis is negligible. The rate of atmospheric photooxidation is moderate. Acetyl tributyl citrate is expected to have low persistence (PI) and low bioaccumulation potential (Bl). The environmental fate properties of acetyl tributyl citrate are provided in Table 2. Conclusion: Acetyl tributyl citrate is a liquid with moderate vapor pressure and moderate water solubility. It is expected to have low mobility in soil. Volatilization is expected to be high, but adsorption to soil and sediment may attenuate the rate of volatilization. The rate of hydrolysis is negligible. The rate of atmospheric photooxidation is moderate. Acetyl tributyl citrate is readily biodegradable. CASRN 77-90-7 is expected to have low persistence (PI) and low bioaccumulation potential (Bl). 5 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Table 2. Environmental Fate Properties of CASRN 77-90-71 Property Value CASRN 77-90-7 Photodegradation Half-life 8.9 hours (estimated)2 Hydrolysis Half-life 139 days at pH 8 (estimated)2; 3.8 years at pH 7 (estimated)2 Biodegradation 26% after 21 days; >90% after 5 hours; 72.9%) after 42 days; 37%o after 45 days in compost soil; 82%o after 28 days (readily biodegradable)3 Bioaccumulation Factor BAF = 12.5 (estimated)2 Log Koc 4.9 (estimated)2 Fugacity (Level III Model)2 Air (%) Water (%) Soil (%) Sediment (%) 2.8 14.8 61.6 20.9 Persistence4 PI (low) Bi oaccumul ati on4 Bl (low) 'The Moiflcx Inc. 2003. Test Plan and Robust Summary for Acetyltributyl citrate. Available online at http://www.epa.gov/chemrtk/pubs/summaries/acetlcit/cl5025tc.htm as of April 30, 2012. 2U.S. EPA. 2012. Estimation Programs Interface Suite™ for Microsoft® Windows, v4.10. U.S. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as of April 30, 2012. 3National Institute of Technology and Evaluation. 2002. Biodegradation and Bioaccumulation of the Existing Chemical Substances under the Chemical Substances Control Law. Available online at http://www.safe.nite.go.ip/english/kizon/KIZON start hazkizon.html as of April 30, 2012. 4Federal Register. 1999. Category for Persistent, Bioaccumulative, and Toxic New Chemical Substances. Federal Register 64, Number 213 (November 4, 1999) pp. 60194-60204. 3. Human Health Hazard A summary of health effects data submitted for SIDS endpoints is provided in Table 3. Acute Oral Toxicity (1) Five rats (strain and gender not reported) were administered acetyl tributyl citrate via gavage (vehicle not specified) at 10 - 30 mL/kg (approximately 10,500 - 31,500 mg/kg) and observed for 21 days. No mortalities were reported. LDso > ~ 31,500 mg/kg 6 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 (2) Four fasted cats (strain and gender not reported) were administered acetyl tributyl citrate by gavage (vehicle not specified) at 30 - 50 mL/kg (approximately 31,500 - 52,500 mg/kg). Additionally, two cats received 50 mL/kg (approximately 52,500 mg/kg) and two cats were administered a control substance (control not identified). The animals were observed for periods of up to 2 months. No mortalities were reported. LDso > ~ 52,460 mg/kg Repeated-Dose Toxicity (1) Sprague-Dawley rats (20/sex/group) were administered acetyl tributyl citrate (purity >98%) via the diet at 0, 100, 300 or 1000 mg/kg-bw/day for 13 weeks. Dose levels were based on the results of a 14-day range-finding study in which increased cytoplasmic eosinophilia was observed in periportal hepatocytes of males and females exposed to 5000 mg/kg-bw/day. Actual doses received in the 13-week study were 0, 101, 302 and 996 mg/kg-bw/day for males and 0, 100, 296 and 999 mg/kg-bw/day for females. Blood was collected on day 85 for comprehensive hematology and clinical chemistry evaluations (including activities of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and gamma glutamyl transferase). Urine samples were collected on days 86 - 87 for comprehensive urinalysis. All rats were killed in week 14 and examined macroscopically. A comprehensive set of tissues (including male and female reproductive tissues) were prepared from all rats. All tissues from control and 1000 mg/kg-bw/day rats were microscopically examined; kidney, liver, lung and gross lesions were examined for all 100 and 300 mg/kg-bw/day rats. The following organs were weighed for all rats: adrenals, brain, heart, kidneys, liver, ovaries, spleen, testes, thymus and thyroid with parathyroids. All rats survived to study termination and no clinical signs were reported. Mean body weights were slightly reduced in males and females at 1000 mg/kg-bw/day and in females at 300 mg/kg-bw/day (statistical significance not reported). Semiquantitative observations revealed a decreased urinary pH in males at 1000 mg/kg-bw/day and crystals in the urine of males at > 300 mg/kg-bw/day and in females at 1000 mg/kg-bw/day. Males exhibited increased serum alkaline phosphatase activity at 1000 mg/kg-bw/day and females exhibited decreased fasting glucose at > 300 mg/kg-bw/day and decreased alanine aminotransferase activity and bilirubin concentration at 1000 mg/kg-bw/day. Males exhibited increased relative liver weights at > 300 mg/kg-bw/day and increased relative kidney weights at 1000 mg/kg-bw/day. Females exhibited increased relative liver weights at 1000 mg/kg-bw/day. No other treatment-related effects were reported. LOAEL = 1000 mg/kg-bw/day (based on decreased body weight and organ weight changes) NOAEL = 300 mg/kg-bw/day (2) In a combined repeated-dose/reproductive/developmental toxicity study, Han Wistar rats (F0; 25/sex/group) were administered acetyl tributyl citrate (purity 99.9%) via the diet at 0, 100, 300 or 1000 mg/kg-bw/day for 4 weeks prior to mating, through mating (F0 males) and through gestation and lactation (F0 females). Actual doses received in the study were 0, 103, 306, and 1013 mg/kg-bw/day for males and 0, 102, 306, and 1024 mg/kg-bw/day for females. The F1 offspring received indirect exposure in utero and from birth until the start of a 13-week toxicity study in which selected F1 offspring (20/sex/group) received dietary doses of 0, 100, 300 or 1000 mg/kg-bw/day for 13 weeks. Additional rats (10/sex/group) were assigned to the high-dose 7 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 group and the control group for inclusion in a 4-week recovery period following the 13-week treatment period. Endpoints included organ and body weights, reproductive performance variables and gross and microscopic histopathology of tissues, which were not specified in the robust summary. Treatment at 1000 mg/kg-bw/day for 13 weeks resulted in a slight decrease in body weight gain, increased liver weights and hepatic hypertrophy and hepatic peroxisome proliferation in both F1 sexes. Weak peroxisome proliferation was measured in males at 300 mg/kg-bw/day. Slight variations in urine composition and plasma electrolyte composition (indicative of an effect on renal function) were seen at the two highest doses, but the effects were within historical control ranges, were reversible and no histopathological changes in the kidneys were seen. No other exposure-related systemic effects (i.e., non-reproductive effects) were mentioned in the robust summary. LOAEL = 1000 mg/kg-bw/day (based on organ weight changes and histopathology in F1 animals) NOAEL = 300 mg/kg-bw/day (3) Rats (4/concentration, mixed male and female; strain not reported) were administered acetyl tributyl citrate via the diet at 5 or 10% (approximately 2500 and 5000 mg/kg-bw/day) for 8 weeks. A control group received basic diet only. Blood was collected for hematology after 4 and 8 weeks of exposure. After exposure, all rats were necropsied and the following tissues were examined microscopically: heart, lungs, gastrointestinal tract, liver, pancreas, spleen and kidneys. No clinical signs were reported. At the end of 8 weeks, no exposure-related effects were found on hematological or histological endpoints. NOAEL ~ 5000 mg/kg-bw/day (highest dose tested) (4) Rats (4/concentration, mixed male and female; strain not reported) were administered acetyl tributyl citrate via the diet at 5 or 10% (approximately 2500 and 5000 mg/kg-bw/day) for 6 weeks. A control group received basic diet only. Body weights were measured initially and weekly until study completion. Diarrhea was observed in animals receiving the 10% acetyl tributyl citrate diet. These animals also exhibited reduced body weight gain (statistics not done due to small sample size). LOAEL -5000 mg/kg-bw/day (based on reduced body weight gain and clinical signs) NOAEL -2500 mg/kg-bw/day (5) Sherman rats (20/group; gender not provided) were administered acetyl tributyl citrate (purity 99.4%) via the diet at 0, 200, 2000 or 20,000 ppm (approximately 0, 10, 100 or 1000 mg/kg-bw) for approximately 2 years. A control group of 40 rats received the basal diet. A transient reduction in body weight gain was observed in all three treatment groups; however, the effect was not reproducible when two additional groups of rats were added to the study at 200 and 2000 ppm (approximately 10 and 100 mg/kg-bw/day) for 1 year. Mortalities, including 12 of 60 treated rats and 8 of 40 control rats, were not attributed to treatment. No clinical signs were reported and lymphoid tumors of the pleural and abdominal cavities were seen in both treated and control animals at comparable rates. There were no differences between treated and untreated rats in comparisons of pathological findings, but reporting of experimental details and results was sparse in the original report. The robust summary noted that the NOAEL appears to be 20,000 ppm (approximately 1000 mg/kg-bw/day) and considered a more appropriate and conservative NOAEL to be 2000 ppm (~ 100 mg/kg-bw/day) due to the lack of experimental 8 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 detail in the study report and the pre-GLP timing of the study. [The original report and the robust summary provide insufficient details to reliably designate a NOAEL or LOAEL for hazard identification purposes. This summary is included for information purposes only.] (6) Two cats (gender and strain not reported) were administered acetyl tributyl citrate via gavage (vehicle not specified) at 5 mL/kg (approximately 5246 mg/kg) daily for 8 weeks. A control group was included in the study, but information on the control substance was not provided. The treated cats developed loose stools and lost weight during the study period. There was no change in behavior or appearance and no effect was observed on the urine, blood chemistry (sugar and creatinine) or blood counts. [Due to the small sample size and the use of a single dose, this study summary is included for information purposes only.] Reproductive Toxicity (1) In a 2-generation reproductive toxicity study, Sprague-Dawley rats (30/sex/group) were administered acetyl tributyl citrate via the diet at 0, 100, 300 or 1000 mg/kg-bw/day, beginning at 11 weeks prior to mating for F0 males and continuing through a maximum mating period of 14 days. F0 females began treatment at least 3 weeks prior to mating and continued through mating, gestation and lactation. F1 offspring were in turn exposed to the treated (100, 300 or 1000 mg/kg-bw/day) or untreated diet from weaning, 10 weeks prior to mating and throughout the mating period (maximum 14 days) for males and females. F1 females continued treatment through gestation and lactation. No treatment-related clinical signs were reported for either of the F0 or F1 parents throughout the study. Body weights of the F1 parental males in the 300 and 1000 mg/kg-bw/day groups were consistently lower than controls, while the body weights of the F1 females and F0 parents were largely unaffected by treatment. Reduced water consumption was noted in F0 and F1 parents in the 1000 mg/kg-bw/day group. Mating, gestation and fertility of the F0 and F1 parents were unaffected by treatment. There were no abnormalities seen at necropsy that were considered treatment-related. The body weights of the pups from the 300 and 1000 mg/kg-bw/day groups were slightly lower than controls and the mortality rates in these litters were slightly higher. No other treatment-related effects were seen in the pups. LOAEL (systemic toxicity) = 300 mg/kg-bw/day (based on reduced body weights in F1 males) NOAEL (systemic toxicity) = 100 mg/kg-bw/day NOAEL (reproductive toxicity) = 1000 mg/kg-bw/day (highest dose tested) (2) In the dietary combined repeated-dose/reproductive/developmental toxicity study in Han Wistar rats described previously, no treatment-related clinical signs were observed in the parental animals. No differences compared to controls were seen in estrous cycles, mating performance, fertility, gestation length and parturition. There were no adverse effects on sperm motility, counts or morphology. Litter size, survival and growth were similar in all groups with the exception that litter size and the number of implantations in the 1000 mg/kg-bw/day group were marginally lower than the control group (statistical significance not provided). At the necropsy of parental animals, no findings were considered treatment-related. As reported in the Repeated-Dose Toxicity section above, a 13-week exposure to 1000 mg/kg-bw/day was associated with a slight decrease in body weight gain, increased liver weights, hepatic hypertrophy and hepatic peroxisome proliferation in both F1 sexes. Weak peroxisome proliferation was measured in F1 males at 300 mg/kg-bw/day. 9 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 LOAEL (systemic toxicity) = 300 mg/kg-bw/day (based on hepatic peroxisome proliferation in F1 males) NOAEL (systemic toxicity) = 100 mg/kg-bw/day LOAEL (reproductive toxicity) = 1000 mg/kg-bw/day (based on decreased litter size and decreased number of implantations) NOAEL (reproductive toxicity) = 300 mg/kg-bw/day Developmental Toxicity In the dietary combined repeated-dose/reproductive/developmental toxicity study in Han Wistar rats described previously, no treatment-related clinical signs were observed in the parental animals. No differences compared to controls were seen in estrous cycles, mating performance, fertility, gestation length and parturition. There were no adverse effects on sperm motility, counts or morphology. Litter size, survival and growth were similar in all groups with the exception that litter size and the number of implantations in the 1000 mg/kg-bw/day group were marginally lower than the control group (statistical significance not provided). At the necropsy of parental animals, no findings were considered treatment-related. NOAEL (maternal toxicity) = 1000 mg/kg-bw/day (highest dose tested) LOAEL (developmental toxicity) = 1000 mg/kg-bw/day (based on decreased litter size and decreased number of implantations) NOAEL (developmental toxicity) = 300 mg/kg-bw/day Genetic Toxicity — Gene Mutation In vitro (1) Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to acetyl tributyl citrate (purity 98.55%) in DMSO at concentrations of 50, 158, 500, 1580 or 5000 |ig/plate in the presence and absence of metabolic activation. The assay was conducted in triplicate, with positive and vehicle controls, but the robust summary did not mention whether or not appropriate results for controls were obtained. The test material was not cytotoxic at 5000 |ig/plate and showed no evidence of mutagenic activity with or without S-9 activation in this assay. Acetyl tributyl citrate was not mutagenic in this assay. (2) Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were exposed to acetyl tributyl citrate in DMSO at concentrations of 333, 1000, 3333, 6667 or 10,000 |ig/plate in the presence and absence of metabolic activation. The assay was conducted in triplicate. Positive and negative controls were included, but the robust summary did not mention if appropriate control results were obtained. Acetyl tributyl citrate was not cytotoxic at 10,000 |ig/plate and showed no evidence of mutagenic activity with or without S-9 activation in this assay. Acetyl tributyl citrate was not mutagenic in this assay. 10 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 (3) L5178Y TK+/- mouse lymphoma cells were exposed to acetyl tributyl citrate in DMSO at concentrations of 10, 70, 150 or 230 |ig/mL in the absence of metabolic activation or 200, 270, 340, 410 or 480 |ig/mL in the presence of metabolic activation and assessed for forward mutations at the TK locus. An initial toxicity assay was conducted using concentrations over a range from 0.1 to 5140 jag/m L in the presence and absence of metabolic activation. Solvent and positive controls tested concurrently and responded appropriately. Complete toxicity was seen in an initial test at concentrations >514 |ag/m L for nonactivated cultures and > 1028 |ag/m L for S-9 activated cultures. There was a dose-dependent increase in cytotoxicity for treated cultures, but there was no increase in mutant frequencies either with or without S-9 activation when compared to solvent controls. Acetyl tributyl citrate was not mutagenic in this assay. (4) Chinese hamster ovary (CHO) cells (two cultures/dose level) were exposed to acetyl tributyl citrate (purity 99.02%) at concentrations of 0 (DMSO, solvent control), 25, 50, 100, 200 or 400 |ig/mL in the presence and absence of metabolic activation and evaluated for HGPRT mutations. Positive and negative controls were tested concurrently and responded appropriately. An approximate 3-fold increase in the number of mutants occurred in the second assay at the 200 |ig/mL dose, without metabolic activation (compared with controls), but the response was not dose-related and not replicated in the first assay. Therefore, the test substance was determined to be not mutagenic in this assay. Acetyl tributyl citrate was not mutagenic in this assay. Genetic Toxicity — Chromosomal Aberrations In vitro Rat lymphocytes were initially exposed to acetyl tributyl citrate (purity 98.55%) in DMSO at concentrations of 4, 13.3, 40, 133 or 400 |ig/plate in the presence and absence of metabolic activation to determine the mitotic indices. Based on those results, the concentrations selected for determining the frequencies of chromosomal aberrations were 50, 133 and 400 |ig/mL. One hundred cells/replicate in the treated and negative controls and 50 cells in the positive control were scored for frequencies of chromosomal aberrations. Positive and negative controls were tested concurrently and responded appropriately. Acetyl tributyl citrate did not induce chromosomal aberrations in this assay. Genetic Toxicity — Other In vivo In an unscheduled DNA synthesis assay, groups of five male Han Wistar rats were administered acetyl tributyl citrate via gavage at 0 (solvent control), 800 or 2000 mg/kg in corn oil. Two positive controls were tested concurrently. Animals were sacrificed at either 2 - 4 or 12 - 14 hours after dosing and primary hepatocyte cultures were obtained. Appropriate results were seen in the positive and negative controls. Acetyl tributyl citrate did not induce unscheduled DNA synthesis. Conclusion: Acute oral toxicity of acetyl tributyl citrate in cats and rats is low. In a 90-day repeated-dose oral dietary study in rats, decreased body weight and organ weight changes were 11 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 observed at 1000 mg/kg-bw/day; the NOAEL is 300 mg/kg-bw/day. In a combined repeated- dose/reproductive/developmental toxicity study in rats, organ weight and histopathological changes were observed in adults at 1000 mg/kg-bw/day; the NOAEL is 300 mg/kg-bw/day. In a 2-generation reproductive toxicity study in rats, reduced body weight was observed in Flmales at 300 mg/kg-bw/day; the NOAEL for systemic toxicity is 100 mg/kg-bw/day. In the same study, no other treatment related effects were observed; the NOAEL for reproductive toxicity is 1000 mg/kg-bw/day, the highest dose tested. In the combined repeated- dose/reproductive/developmental toxicity study in rats previously described, histopathological changes were observed in the liver of adult males at 300 mg/kg-bw/day; the NOAEL for systemic toxicity is 100 mg/kg-bw/day. In the same study, decreased litter size and decreased number of implantations were observed at 1000 mg/kg-bw/day; the NOAEL for reproductive and developmental toxicity is 300 mg/kg-bw/day. The NOAEL for maternal toxicity is 1000 mg/kg- bw/day (highest dose tested). Acetyl tributyl citrate did not induce gene mutations in bacteria or mammalian cells in vitro and did not induce chromosomal aberrations in mammalian cells in vitro. Table 3. Summary of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Human Health Data Endpoint Acetyl Tributyl Citrate (77-90-7) Acute Oral Toxicity LDso (mg/kg) (rats) >~ 31,500 (cats) >-52,460 Repeated-Dose Toxicity NOAEL/LOAEL Oral Diet (mg/kg-bw/day) (rat, 90-day) NOAEL = 300 LOAEL = 1000 Reproductive Toxicity Oral Diet (mg/kg-bw/day) Reproductive Toxicity (rat) NOAEL = 300 LOAEL =1000 (rat; 2-gen) NOAEL = 1000 (highest dose tested) 12 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Table 3. Summary of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Human Health Data Endpoint Acetyl Tributyl Citrate (77-90-7) Developmental Toxicity Oral Diet (mg/kg-bw/day) Maternal Toxicity (rat) NOAEL = 1000 (highest dose tested) Developmental Toxicity NOAEL = 300 LOAEL =1000 Genetic Toxicity - Gene Mutations in vitro Negative Genetic Toxicity - Chromosomal Aberrations in vitro Negative Genetic Toxicity - Unscheduled DNA synthesis Negative 4. Hazard to the Environment A summary of aquatic toxicity data submitted for SIDS endpoints is provided in Table 4. Acute Toxicity to Fish (1) Bluegill sunfish (Lepomis macrochirus) were exposed to acetyl tributyl citrate at nominal concentrations up to 120 mg/L under flow-through conditions for 96 hours, followed by a 96- hour observation period. Measured concentrations were not provided in the original report. 96-h LC50 = 38-60 mg/L (2) Mummichogs (.Fundalus heteroclitus) were exposed to acetyl tributyl citrate at nominal concentrations up to 120 mg/L under flow-through conditions for 96 hours, followed by a 96- hour observation period. Measured concentrations were not provided in the original report. Flow-rate, pH, dissolved oxygen concentration and temperature were monitored. 96-h LC50 = 59 mg/L Acute Toxicity to Aquatic Invertebrates Water fleas (Ceriodaphnia dubia) were exposed to acetyl tributyl citrate at nominal concentrations of 0 (control), 3.9, 7.8, 16.0, 31, 63 or 125 mg/L under static conditions for 48 13 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 hours and assessed for immobilization. Mean measured concentrations were: less than the detection limit (control), 3.82, 4.82, 8.70, 15.5, 17.9 or 60.2 mg/L. 48-h ECso=7.82 mg/L Toxicity to Aquatic Plants Green algae (Desmodesmus subspicatus) were exposed to tributyl acetyl citrate at nominal concentrations of 10, 20, 40, 80, and 100 mg/L under static conditions for 72 hours for algae growth inhibition test. The mean measured concentrations (arithmetic mean) of the aqueous phase (bioavailable fraction) were for the evaluation: 4.65, 10.9, 12.9, 44.6 and 47.1 mg/L. The measured concentrations deviated more than 20 % from the nominal values. Therefore, the reported effect concentrations refer to mean measured concentrations (arithmetic mean) of the measured concentrations. 72-h EC50= 74.4 mg/L (growth rate) 72-h LOEC = 10.9 mg/L (growth rate) 72-h NOEC= 4.65 mg/L (growth rate) 72-h ECso= 11.5 mg/L (yield) 72-h LOEC = 10.9 mg/L (yield) 72-h NOEC = 4.5 mg/L (yield) http://apps.echa.europa.eu/registered/data/dossiers/DISS-9d99c2al-cel8-2d95-e044- 00144f67d249/AGGR-2aee6091 -992c-41 fd-80c7-3e0e2740a67b PISS-9d99c2al -ce 18-2d95- e044-00144f67d249. html# AGGR-2aee6091 -992c-41 fd-80c7-3 e0e2740a67b Chronic Toxicity to Aquatic Invertebrates Water fleas (Daphnia magna) were exposed to acetyl tributyl citrate at nominal concentration of 1.5 mg/L under semi-static conditions for 21 days and assessed for survival and reproduction. The 21-day NOEC was determined to be > 1.11 mg test item/L for the reproduction rate and > 1.11 mg test item/L for the survival of the adult test animals, respectively. The EC50 for the reproduction rate was >1.11 mg test item/L (effect value based on analytical monitoring - TWA) 21-d ECso > 1.11 mg/L (survival) 21-d EC50 > 1.11 mg/L (reproduction) http://apps.echa.europa.eu/registered/data/dossiers/DISS-9d99c2al-cel8-2d95-e044- 00144f67d249/AGGR-0el30278-8aab-4c90-ble8-418778935e93 DISS-9d99c2al-cel8-2d95- e044-00144f67d249.html#AGGR-0e 13 0278-8aab-4c90-b 1 e8-41877893 5e93 Conclusion: For acetyl tributyl citrate, the 96-h LC50 values for fish range from 38 to 60 mg/L, the 48-h EC50 value for aquatic invertebrates is 7.8 mg/L and the 72-h EC50 values for aquatic plants are 11.5 mg/L for biomass and 74.4mg/L for growth rate, respectively. The chronic 21-d EC50 values for aquatic invertebrates are > 1.11 mg/L (survival) and >1.11 mg/L (reproduction). 14 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Table 4. Summary of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Aquatic Toxicity Data Endpoint Acetyl Tributyl Citrate (77-90-7) Fish 96-h LCso (mg/L) 38-60 Aquatic Invertebrates 48-h ECso (mg/L) 7.8 Aquatic Plants 72-h ECso (mg/L) (growth rate) (biomass) 74.4 11.5 Chronic Toxicity to Aquatic Invertebrates 21-d ECso (mg/L) >1.11 (survival) >1.11 (reproduction) (Bold) = experimentally derived data (i.e., derived from testing) 15 ------- |